Up to 99% of cervical cancer and 90% of oropharyngeal cancer cases are caused by Human Papillomavirus (HPV) infections. Although two HPV vaccines are in clinical use for the prevention of HPV- associated-cancers, there are limitations in the scope and worldwide cost-effectiveness of the current vaccines. In particular, while the vaccines may be prophylactic, they are not likely to be therapeutic. Moreover, the vaccines are only effective against four HPV types. Therefore, it is important to develop novel anti-viral therapies that can augment the efficacy of the current available therapies to prevent and treat HPV infection. One strategy is to develop drugs that target specific steps of the HPV life cycle. However, knowledge of the HPV infectious trafficking pathway is incomplete, and further research is required to more clearly define this pathway in order to identify novel drug targets. Evidence suggests that after the virus enters the cell, it traffics from the early endosom to Golgi. In support of this, it was recently shown that the retromer, a major cellular trafficking complex involved in endosome to Golgi transport, is crucial for HPV infection. Specifically, the viral minor capsid protein L2, within an endosomal vesicle, is able to directly interact with the cytoplasmic retromer complex in order to traffic from the endosome to the Golgi. Exactly how virus located within the endosome is able to recognize the retromer complex in the cytoplasm is currently unknown. This proposal seeks to address the mechanism by which HPV capsid proteins penetrate the endosomal membrane for gaining access to the retromer. To address this goal, I will identify HPV mutants that accumulate in the endosome and thereby determine the viral capsid protein sequences necessary for endosomal membrane penetration. Microscopy and biochemical techniques will be used to assess capsid protein penetration of the endosome. Overall, this study should provide further insight into the HPV trafficking pathway and thus aid in the development of anti-viral therapeutics for the prevention of HPV-associated cancers.